title: Spatial filtering nearly eliminates the side-lobes in single- and multi-photon 4pi-type-C super-resolution fluorescence microscopy.
abstract: Super-resolution microscopy has tremendously progressed our understanding of cellular biophysics and biochemistry. Specifically, 4pi fluorescence microscopy technique stands out because of its axial super-resolution capability. All types of 4pi-microscopy techniques work well in conjugation with deconvolution techniques to get rid of artifacts due to side-lobes. In this regard, we propose a technique based on spatial filter in a 4pi-type-C confocal setup to get rid of these artifacts. Using a special spatial filter, we have reduced the depth-of-focus. Interference of two similar depth-of-focus beams in a 4π geometry result in substantial reduction of side-lobes. Studies show a reduction of side-lobes by 46% and 76% for single and two photon variant compared to 4pi - type - C confocal system. This is incredible considering the resolving capability of the existing 4pi - type - C confocal microscopy. Moreover, the main lobe is found to be 150 nm for the proposed spatial filtering technique as compared to 690 nm of the state-of-art confocal system. Reconstruction of experimentally obtained 2PE - 4pi data of green fluorescent protein (GFP)-tagged mitocondrial network shows near elimination of artifacts arising out of side-lobes. Proposed technique may find interesting application in fluorescence microscopy, nano-lithography, and cell biology.
authors.affiliation: Nanobioimaging Laboratory, Department of Instrumentation and Applied Physics, Indian Institute of Science, Bangalore 560012, India.
authors.forename: Kavya
authors.initials: K
authors.lastname: M
authors.forename: Raju
authors.initials: R
authors.lastname: Regmi
authors.forename: Partha P
authors.initials: PP
authors.lastname: Mondal
mesh_terms: D036641:Microscopy, Fluorescence, Multiphoton / Q000295:instrumentation* / Q000379:methods*; D008962:Models, Theoretical